GB1566014A - Test for barbiturates in body fluid outside the human body and reagents useful therefor - Google Patents

Description

PATENT SPECIFICATION

( 11) ( 21) Application No 22062/77 ( 22) Filed 25 May 1977 ( 31) Convention Application No.

690 123 ( 32) Filed 26 May 1976 in ( 33) United States of America (US) ( 44) Complete Specification published 30 April 1980 ( 51) INT CL 3 C 07 D 239162 11 GOIN 33/54 ( 52) Index at acceptance C 2 C 1603 200 215 220 22 Y 247 250 252 25 Y 282 30 Y 321 32 Y 332 342 34 Y 351 352 366 367 368 387 43 X 490 491 584 620 621 628 62 X 645 658 65 X 660 661 66 X 761 768 AA KS LZ MM TW C 3 J AS G 1 B BR 1 566014 ( 19 ( 54) A TEST FOR BARBITURATES IN BODY FLUID, OUTSIDE THE HUMAN BODY, AND REAGENTS USEFUL THEREFOR ( 71) We, F HOFFMAN-LA Roc HE & Co., AKTIENGESFLLSCH Ai FT, a Swiss Company of 124-184 Grenzacherstrasse, Basle, Switzerland, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:

The present invention relates to a test for barbiturates in body fluid, outside the human body, and to novel reagents which are useful in a sensitive diagnostic test to detect the presence of barbiturates in body fluids, outside the human body.

The large increase in the abuse of therapeutic agents, particularly the barbituates, by the general population as well as by military personnel, has brought with it a substantial need to improve analytical techniques for the determination of such agents in biological fluids In many instances, medical treatment centres are faced with the immediate need for determining the identity of a barbituate taken by a patient who is unable, being in a comatose condition, or unwilling to supply such information to the physician carrying out the treatment Early procedures involved the identification of barbiturates by extraction and thin-layer gas chromatographic and spectrophotometric methods These procedures have the disadvantages of being relatively time-consuming and laborious and lacking great sensitivity Recently, a rapid and sensitive immunoassay procedure involving the reaction between antibodies and barbiturate antigen has been described by S Spector in United States Patent No 3766 162 and by S Spector and E J Flynn in Science, 174, 1037 ( 1971).

This procedure, however, requires sophisticated and expensive equipment such as scintillation counters Therefore, it would be desirable to develop a rapid and highly sensitive assay for detecting the presence of barbiturates in body fluids, outside the human body, which would not require sophisticated equipment and could be easily carried out by laboratory technicians having a minimum of training.

The present invention is concerned with 50 a novel class of barbiturate derivatives, namely, aminoaryl esters and amino-(lower alkyl) amides of carboxy-substituted barbiturates, which esters and amides may be covalently coupled via an amide linkage to 55 a carboxylated latex polymer The barbiturate thus-linked to the latex polymer by means of the linking group can then be used as a reagent in a sensitive diagnostic assay for the presence of barbiturates in body 60 fluids, outside the human body This assay method is dependent upon the well-known binding of antigen to antibodies specific therefor, which is manifested by an insolubilisation or agglutination followed by 65 flocculation When either the antigen or the antibody is linked to a suitable polymer such as a latex polymer, as hereinafter described, the detection of the antigen-antibody binding by means of agglutination is significantly en 70 hanced by means of the latex so that such agglutination reaction is easily seen by the naked eye.

The general technique of using latex particles as carriers for antigens or antibodies 75 for easy visulisation of the antigen-antibody reaction has been previously described in the literature (e g United States Patent Specification No 3 857 931).

The starting materials which are used for 80 the preparation of the latex reagents provided by the present invention are aminoaryl esters and amino (lower alkyl) amides of carboxy-substituted barbiturates As used herein, the term "lower alkyl" refers to 85 straight-chain and branched-chain saturated hydrocarbon groups containing from 2 to 8 carbon atoms inclusive, such as ethyl, propyl, n-butyl and isobutyl The term "aryl" denotes an aromatic radical derived from an 90 2 1566014 unsubstituted or substituted arene and includes phenyl, naphthyl, halophenyl, tolyl, anisyl nitrophenyl and hydroxyphenyl The term "halide" denotes iodide, bromide and chloride.

The barbiturates useful for binding to latex polymers are those having free carboxylic acid groups A particularly preferred barbiturate for use as a starting material in connection with the present invention is 5-allyl-5 (l-carboxy-isopropyl) barbituric acid (allonalcarboxylic acid) since it has a carboxylic acid group in the side-chain and is readily obtainable Thus, particularly preferred reagents are aminoaryl esters and amino-(lower alkyl) amides of the carboxylic acid group of 5-propyl-5-( 1-carboxy-isopropyl)-barbituric acid However, the present assay, as hereinafter described, will detect barbiturates with or without free or functionalised carboxylic acid groups such as barbital, phenobarbital, amobarbital, pentobarbital and butabarbital.

The aminoaryl esters and amino-(lower alkyl) amides of carboxy-substituted barbiturates, as described earlier, are conveniently prepared from carboxy-substituted barbiturates The requisite aminoaryl or amino(lower alkyl) moiety can conveniently be introduced according to well-known methods.

Thus, for the preparation of aminoaryl esters of carboxy-substituted barbiturates using allonalcarboxylic acid as a starting material, for example, said acid can be esterified with, for example, p-nitrophenol to give 5-allyl-5( 1 p nitrophenyloxycarbonyl isopropyl}barbituric acid which can then be reduced to give 5-propyl-5-(p-aminophenyloxycarbonyl-isopropyl)-barbituric acid.

The esterification is carried out in the presence of a condensing agent dissolved in an inert organic solvent Suitable condensing agents include carbodiimides such as NN'diphenylcarbodiimide and N,N'-dicyclohexylcarbodiimide Suitable inert organic solvents include polar aprotic solvents such as N,N-dimethylformamide, dimethylsulphoxide and hexamethylphosphoramide alone or admixed with non-polar aprotic solvents such as acetone, acetbnitrile and ethyl acetate A particularly preferred condensing agent is N,N'-dicyclohexylcarbodiimide and a particularly preferred organic solvent system is N,N-dimethylformamide/ ethyl acetate.

The temperature at which the esterification is carried out is not narrowly critical The esterification may be carried out at a temperature between O C and 50 C, most preferably at O C to 25 C.

The reduction is carried out by treating the thus-obtained nitroester, dissolved in a suitable inert organic solvent, preferably an alkanol such as methanol, ethanol or 2propanol, with hydrogen in the presence of a suitable hydrogenation catalyst until the uptake of hydrogen ceases Included among the suitable hydrogenation catalysts which can be used are platinum, palladium, rhodium, ruthenium and nickel, unsupported 70 or supported on carriers such as carbon, silica and alumina A particularly preferred hydrogenation catalyst is 10 % palladium-oncarbon While the temperature and pressure at which the hydrogenation is carried out 75 are not critical, it is preferred to carry out the hydrogenation at about room temperature and about atmospheric pressure.

For the preparation of amino-(lower alkyl) amides of carboxy-substituted barbiturates 80 using allonalcarboxylic acid as an example, said acid can be aminated with, for example, 1,4-diaminobutane to give 5-allyl-5 l 1-( 4aminobutylcarbamoyl) isopropyll barbituric acid 85 The amination is carried out in the presence of a condensing agent dissolved in an inert organic solvent Suitable condensing agents include carbodiimides such as N,N'diphenylcarbodiimide and N,N'-dicyclohex 90 ylcarbodiimide and carbonyldiimidazoles such as 1,1 'carbonyldiimidazole Suitable inert organic solvents are ethereal solvents such as monoglyme, diglyme, diozane and tetrahydrofuran A particularly preferred con 95 densing agent is 1,1 '-carbonyldiimidazole A particularly preferred inert organic solvent is tetrahydrofuran.

/The temperature at which the animation is carried out is not narrowly critical An 100 amination temperature of from O C to the boiling point of the solvent is preferred with an amination temperature of about 25 C being most preferred.

The carboxy-sutbstituted barbiturates are 105 also conveniently prepared by well-known methods For example, the carboxysubstituted barbiturates can be readily prepared by the reductive alkylation of barbituric acid with an aldehydo or keto-ester 110 followed by alkylation and saponification of the ester group of the resulting 5,5-disubstituted-barbituric acid.

The reductive alkylation is carried out by treating barbituric acid and an aldehydo 115 or keto-ester such as ethyl formylacetate, ethyl acetoacetate or ethyl levulinate with hydrogen in the presence of a metal hydrogenation catalyst to give a 5-monosubstituted-barbituric acid derivative Suitable 120 metal catalyst are nickel and the noble metals such as platinum, palladium, rhodium and ruthenium The catalysts are normally used in finely divided form and may be either unsupported or present on a suitable 125 inert carrier such as carbon, aluminium, silica and calcium carbonate A particularly preferred catalyst is 10 % palladium-oncarbon.

As solvents for the reductive alkylation 130 1566014 1 566 014 there may be mentioned alcohols such as methanol, ethanol and 2-propanol, and esters such as ethyl acetate.

While the reductive alkylation may be carried out over a wide range of temperatures and pressures from, for example, room temperature to 150 C and atmospheric pressure to 68 atmospheres, it is preferred to carry out the reductive alklation at a temi O perature of 90 C to 100 C and a pressure of about 48 atmospheres.

The alkylation is carried out by treating the 5-mono-substituted-barbituric acid derivative with an alkylating agent such as a methyl halide, propyl halide, allyl halide, hexyl halide or cyclohexenyl halide in a suitable inert solvent in the presence of a base to give a 5,5-disubstituted-barbituric acid derivative Suitable solvent include inter alia, alcohols such as methanol, ethanol and 2propanol, water, and mixtures of water and alcohols Suitable bases are alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, alkaline earth metal hydroxides cuch as calmium hydroxide and alkili metal alkoxides such as sodium methoxide, potassium ethoxide and potassium tert butoxide A particularly preferred base and solvent system is about 20 % aqueous sodium hydroxide The alkylation may be carried out at a temperature in the range of from 10 C,to 80 C, most preferably at a temperature between 25 C and 60 C.

The saponification is carried out by treating the 5,5-disubstituted-barbituric acid derivative with an aqueous acid such as hydrochloric acid, hydrobromic acid or dilute sulphuric acid at an elevated temperature It is preferred to carry out the saponification using about 1-N aqueous hydrochloric acid at a temperature of about the reflux temperature of the saponification mixture.

In order to prepare a diagnostic reagent provided by the present invention, an aminoaryl ester or amino-(lower alkyl) amide of a carboxy-substituted barbiturate is covalently bonded by means of an amide linkage to a latex polymer containing carboxyl groups.

Suitable latex polymers for this purpose are carboxylated syrene butadienes, carboxylated polystyrenes and acrylic acid polymers.

Among the commercial latex polymers which are included in the aforementioned classes are Dow 421, Dow 816, Dow 620, Fluka 241 and Dow 421 Dow batch 1721, a latex polymer of the polystyrene type having a particle size of 0 2 to 0 3 microns, a per cent solid composition of 8 to 12 % and a specific gravity of about 1 02, is also suitParticularly preferred polymers are carboxylated styrene butadiene copolymers, preferably Flucka 241 or Dow 421 Suitable latex carrier particles are generally supplied commercially as an aqueous latex suspension, usually in concentrations of 5 to 60 o solids These polymers are water-insoluble, have a particle size in the range from 0 01 70 to 0 9 microns, preferably between 0 1 and 0.3 microns, and a specific gravity near that of water enabling them to remain in aqueous suspension The particles should have sufficient surface charge density so that, when 75 coupled to the aminoaryl esters and amino(lower alkyl) amides of carboxy-substituted barbiturates, their repulsive forces are sufficient to prevent aggregation.

The aminoaryl esters and amino-(lower 80 alkyl) amides of carboxy-substituted barbiturates are coupled to the carboxylated latex polymers by means of an amide linkage initiated in the presence of a water-soluble carbodiimide condensing agent The degree 85 of coupling is dependent upon the density of the reactive groups in the polymer The density of the reactive groups is not critical to the operability of this invention, provided that a sufficient number of reactive groups 90 are present to provide coupling of a sufficient amount of barbiturate moiety to be useful in a diagnostic test However, a suitable density would be in the range of from 1 to 5,oi preferably about 3 %, by weight The coup 95 ling reaction with carbodiimides is described in detail in United States Patent Specification No 3 857 931.

Once the latex coupled product is formed, it can be used in specific diagnostic tests 100 for the detection of barbiturates It can be used in any convenient concentration depending upon the specific test and samples involved However, concentrations of from 0.1 to 2 % by weight of latex solids are suit 105 able and the preferred concentrations are from 0 3 to 1 5 % by weight.

In a typical test, a measured amount of antiserum against barbiturates is mixed with a barbiturate-free body fluid such as serum, 110 saliva or urine Then, a measured amount of aminoaryl or amino-(lower alkyl) barbiturate coupled latex is added and the mixture is allowed to incubate at a slightly elevated temperature (e g 37 C) for a period of time 115 (e.g from 1 to 3 hours, preferably for about 2 hours) The p H of the test mixture is suitably in the range of from p H 5 0 to 8 5, most preferably 6 5 to 7 5 After the incubation, flocculation or agglutination of the 120 latex particles is noted The concentration and quantity of both the antiserum and the latex complex are adjusted to produce a strong flocculation and the minimum concentrations of both reagents which produce 125 a strong flocculation are determined The mixture of antiserum against barbiturate and barbiturate-free body fluid may be incubated at a slightly elevated temperature (e g 37 C) prior to the addition of the aminoaryl or 130 1 566 014 amino-(lower alkyl) barbiturate coupled latex.

The antisera which may be used in the present diagnostic test are antisera specific for barbiturates such as secobarbital and pentobarbita I The preparation of such antisera is described in United States Patent Specification No 3 766 162 and in Science,

174, 1037 ( 1971).

After the control system has been set up, as described earlier, various amounts of barbiturates (e g secobarbital, pentobarbital, butabarbital, amobarbital, phenobarbital and barbital) are dissolved in barbiturate-free body fluid The minimum amount of barbiturate required to inhibit the flocculation is noted This quantity will depend both upon the concentration and the amount of body fluid added, as well as upon the concentration and the strength of the antiserum used in the test.

In one preferred test, the quantities and concentrations are adjusted so that 400-500 microlitres of serum or urine containing between 100 and 200 nanograms of barbiturate per millilitre (a total of between 40-50 and 80-100 nanograms of barbiturate) will be just sufficient to inhibit flocculation Once the test has been standardised with one type of body fluid (e g urine), another type of body fluid (e g serum) should not be substituted, and a separate standard must be set up for this.

Since the persence of flocculation is easily seen by the naked eye, the presesnt test serves as an extremely sensitive assay method for the detection of barbiturates such as secobarbital, pentobarbital, butabarbital, amobarbital, phenobarbital and barbital Thus, once the test has been standardised as mentioned earlier, the presence of nanogram quantities of these barbiturates in body fluids can easily be detected by noting the inhibition of flocculation caused by the presence of such barbiturates in the body fluid, as compared with the flocculation resulting when barbiturate-free body fluid is used.

The test can be standardised so that a medically and statistically meaningful cutoff point is established Thus, quantities of barbiturates in body fluid greater than this amount will cause inhibition of flocculation (a positive test for the pressence of such drug in the body fluid) and quantities less than this amount will not inhibit flocculation (a negative test).

The reagents described earlier can be conveniently packaged for commercial purposes; for example, in a diagnostic reagent kit containing two separate containers, one having the antiserum against barbiturates and the other having the aminoaryl esters or amino(lower alkyl) amides of carboxy-substituted barbiturates bonded via an amide linkage to latex particles containing carboxyl groups, most preferably in aqueous suspension.

In the Examples 1 'to 9 which are given below with the intention of facilitating an appreciation of the present invention, Ex 70 amples 1 to 4 are concerned with preliminary preparative procedures, Examples 5 and 6 illustrate the invention by describing the preparation and properties of certain sub.

stituted barbituric acids claimed herein, Ex, 75 ample 7 illustrates the invention by describing the,preparation of certain latex polymer diagnostic reagents claimed herein, Example 8 is concerned with a diluted antiserum for testing purposes, and Example 9 is con 80 cerned with a barbiturate test method, Example'1

A mixture of 250 g of barbituric acid, 215 g of ethyl acetoacetate, 10 g of 10 % palladiumon-carbon and 400 ml of methanol was 85 placed in a 2 litre glass-lined reaction vessel and hydrogenated at 90 -100 C and 48 atmospheres for 20 hours 2 litres of water were added to the suspension and the mixture was heated to bring about solution The 90 catalyst was removed by filtration and the filtrate was cooled overnight The crystals were collected to yield 209 g of 5-( 1-ethoxycarbonyl-isopropyl)-barbituric acid of melting point 160 -162 C The mother liquor 95 was evaporated in vacuo and the residue was supended, with stirring, in a small volume of boiling ethyl acetate The solution was allowed to cool to room temperature and unreacted barbituric acid was collected 100 An additional 65 2 g (total yield 87 6 % based on recovered barbituric acid) of the ester were obtained from the filtrate It had a melting point of 160 -162 C.

Example 2 105

In a 1 litre three-neck flask equipped with a mechanical stirrer, reflux condenser and dropping funnel were placed 100 g of 5-( 1ethoxycarbonyl-isopropyl)-barbituric acid (of Example 1), 100 mg of calcium sulphate, 110 mg of copper dust and 500 ml of water.

The mixture was stirred at room temperature for 15 minutes and 53 6 g of allyl bromide were added in one portion followed by the dropwise addition of 125 ml of 20 % sodium 115 hydroxide over 45 minutes at 50 -55 C.

After stirring at 50 -55 C for 3 hours, the mixture was cooled to about 30 C and an additional 26 8 g of allyl bromide were added in one portion The mixture was 120 heated with stirring for 90 minutes and a solution ( 65-70 ml) of 20 % sodium hydroxide was then added at a rate such that the mixture was always slightly alkaline The mixture was cooled to room temperature The p H of 125 the mixture was adjusted to about 9 by the addition of 20 % sodium hydroxide and the mixture was extracted with four 250 ml portions of ethyl acetate The combined ethyl acetate extracts were re-extracted with four 130 1 566 014 300 ml portions of 1 % sodium hydroxide and the aqueous extracts were added to a mixture of 500 g of ice and 300 ml of 6-N hydrochloric acid The precipitate which formed upon standing at room temperature overnight was collected to give 91 0 g ( 78 %) of 5-allyl-5-( 1-ethoxycarbonyl-isopropyl)barbituric acid of melting point 112 -114 C.

Example 3

A mixture of 50 g of 5-allyl-5-(l-ethoxycarbonyl-iso-popyl)barbituric acid (of Example 2) and 400 ml of 1-N hydrochloric acid was heated under reflux for 4 hours The solution was allowed to stand at room ternmperature overnight and the precipitate was collected to yield 42 5 g ( 94 %) of 5-allyl-5(l-carboxy-isopropyl)-barbituric acid of melting point 202 -203 C Example 4

A solution of 2 19 g of ethyl chloroformate in 90 ml of chloroform was added to a solution of 5 08 g of allonalcarboxylic acid l 5-ailyl-5 (l-carboxy-isopropyl) -barbituric acidl (of Example 3) and 2 02 g of triethylamine in 100 ml of dichloromethane The mixture was cooled to 005 C and; stirred at room temperature for 5 hours 3 06 g of pnitrophenol were added and the mixture was stirred at room temperature for 14 hours.

The mixture was washed with three 25 ml portions of saturated sodium carbonate solution and water until neutral, dried over anhydrous sodium sulphate and filtered Evapoation of the filtrate under reduced pressure gave 4 7 g of an oil Crystallisation from ether/heptane ( 1:1) gave 2 2 g of 5allyl-5 (l-p-nitrophenyloxycarbonyl-isopropyl)-barbituric acid of melting point 140 1410 C.

Example S

A solution of 750 g of 5-alilyl-5-(p-aminophenyloxycarbonyl isopropyl) barbituric acid in 15 in of ethanol containing 100 mg of 10 % palladium-on-cwrbon as catalyst was subjected to hydrogenation at room temperature and atmospheric pressure After 1 hour, the required amount ( 180 ml) of hydrogen had been taken up The mixture was filtered through Celite and the filtrate was evaporated to dryness (The word' "Celite" is a registered Trade Mark) The residue was chromatographed on 13 g of silica gel with ethyl acetate as the eluent Fractions containing the desired product were evaporated.

The remaining solid was recrystallised from chloroform/ether to give 620 mg ( 89 %) of 5-propyl-5-(p-aminohenyloxycarbonyl-isopropyl)-barbituric acid of melting point 212 -215 C.

Example 6

A mixture of 2 5 g of 5-allyl-5-( 1-carboxyisopropyl)-barbituric acid (of Example 3), 1.95 g of 1,1 '-carbonyldiimidazole and 100 m I of tetrahydrofuran was stirred under nitrogen at room temperature for 3 hours.

To the solution were added 4 5 g of 1,4diamino-butane and the mixture was stirred overnight The gummy solids were decanted and suspended in hot tetrahydrofuran The crystalline material was collected to yield 1 8 70 g of 5-alhlyl-5-l 1-( 4-aminobutylcarbamoyl)isopropylll-barbituric acid of melting point -165 C A sample reorystallised from ethanol melted at 170 C.

5-Allyl-5-l 1 ( 3-aminopropylcarbamoyl) 75 isopropyll barbituric acid was prepared from 2.5 g of 5-allyl-5-( 1-carboxy-isopropyl)-barbituric acid, 2 g of 1,1 '-carbonyldiimidazole, ml of tetrahydrofuran and 4 g of 1,3diaminopropane by the foregoing procedure 80 The product melted at 202 -203 C.

Example 7

Preparation of latex polymers of amrnoaryl esters and amnino-(lower alkyl) amides of carboxy-substituted barbiturates general 85 procedure 5-Allyl-5 l 1 ( 4-aminobutylcarbamoyl) isopropyll-barbituric acid (of Example 6, paragraph 1) is reacted with a latex suspension in the presence of l-cyclohexyl-3-( 2 90 morpholino-ethyl) carbodiimide metho-ptoluene-sulphonate or 1-ethyl-3-( 3-dimethylaminopropyl)-carbodiimide hydrochloride.

The resulting complex is washed by repeated sedimentation of the solid latex particles by 95 centrifugation and resuspension in an appropriate buffer until no free aminobutyl-barbituric acid is left in the aqueous phase of the suspension The aminobutylbarbituric acid is used at a concentration of 1 mg per 100 ml in distilled water The solution is adjusted to p H 5 0 The latex suspension is a carboxylated styrene butadiene copolymer (Fluka No 241) which has been washed and then diluted in water to contain approxi 105 mately 8 % latex solids The concentration of the carbodiimide used is 1 % weight-volume in distilled water The mixture is comprised of the following ratio of reactants: one volume of 1 % carbodiimide, one 110 volume of amino-butylbarbituric acid and three volumes of latex polymer suspension.

The reaction is allowed to proceed at room temperature for 2-16 hours under continuous agitation The solid polymer complex 115 is then sedimented by centrifugation, washed with water and resuspended in O 1-,3 Tris saline buffer, p H 7 3, to a final concentration of 4 mg solids/ml.

Latexes such as Dow 816, Dow 421 and 120 Dow batch 1721 may be used instead of Fluka No 241 and acetonitrile may be used as the solvent in the coupling reaction when 5-propyl-5 ( 1-p-aminophenyloxycarbonyl isapropyl)-barbituric acid (of Example 5) is 125 used as the barbituric acid derivative.

Example 8

Preparation of antiserum for test Rabbit antiserum against barbiturates, prepared as described in United States 130 $ 61 566 014 Patent Specification No 3 766 162, is diluted in an appropriate buffer system This diluent consists of the following in aqueous solution at p H 7 3:

1 0 01 % Thimerosal 2 1 % Normal rabbit serum 3 0 01 % EDTA lethylenediamine tetraacetate in the disodium forml 4 Tris l O 1 M Tris (hydroxymethyl)aminomethane hydrochloridell 0 85 % Sodium chloride.

Example 9

Test method 2 ml of diluted antiserum prepared as described in Example 8 are dispensed into small test tubes ( 10 x 75 mm) To this quantity are added 400-500 microlitres of barbiturate-free urine The two fluids are mixed and left to incubate at 37 C for 10 minutes Ten microlitres of diluted aqueous 5-allyl-5 l 1 ( 4-aminobutylcarbamoyl) -ison propylll-barbituric acid latex suspension containing approximately 0 3 % latex solids by weight are added and mixed with the antiserum and urine The test tubes are then placed in a 37 C water-bath or metal heat block so that approximately 1 5 cm of the liquid column in the test tube is under water or inside the metal heat block The appearance of the liquid in the tubes is translucent, turbid or slightly milky For negative samples, fine floccules are visible in the tube during the first hour Large, easily visible floccules become evident during the second hour of incubation and tend to settle out leaving the liquid increasingly more clear and transparent.

The dilution of a particular antiserum which is chosen for the test system is the one which represents the highest dilution which still produces a strong flocculation after 2 hours, as dencribed earlier When various amounts of barbiturates are dissolved in barbiturate-free normal urine and substituted for the barbiturate-free urine in the test systems, no flocculation occurs The amount of barbiturate required to inhibit the flocculation will usually vary from 100 nanograms per nil or greater depending on the concentration of antiserum used and the strength of the antiserum produced in the donor animal It is also dependent on the amount of body fluid added Thus, for the system described earlier, 400-500 tl of urine containing 200-300 nanograms of barbiturate/ml is just sufficient to inhibit flocculation 100 Al of urine containing 800-1000 nanograms/ml or 50 dl of urine containing 1600-2000 nanograms/ml will behave similarly 60

Claims (17)

WHAT WE CLAIM IS:-

1 An aminoaryl ester or amnino-(lower alkyl) amide of a carboxy-substituted barbiturate.

2 A compound according to claim 1, 65 wherein the barbiturate is 5-propyl-5-( 1carboxy-isopropyl)-barbituric acid.

3 A compound according to claim 1 or claim 2, wherein the aminoaryl ester is an aminophenyl ester 70

4 A compound according to claim 1 or claim 2, wherein the amino-(lower alkyl) amide is an aminobutyl amide.

A compound according to claim 1 or claim 2, wherein the amino-(lower alkyl) 75 amide is an aminopropyl amide.

6 5-Propyl 5 ( 1-p-aminophenyloxycarbonyl-isopropyl)-barbitu ric acid.

7 5-Allyl-5 l 1-( 4-aminobutyl)carbamoylisopropyll-barbituric acid 80

8 5-Allyl-5-l 1-( 3-aminopropyl)carbamoylpropyll-barbituric acid.

9 An immunological diagnostic reagent comprising discrete particles of latex polymer having carboxyl groups covalently bound 85 through an amide linkage to the amino group of an aminoaryl ester or amino-(lower alkyl) amide of a carboxy-substituted barbiturate.

A reagent according to claim 9, wherein the barbiturate is 5-isopropyl-5-( 1 90 carboxy-isopropyl)-barbituric acid.

11 A reagent according to claim 9, wherein the latex polymer is a carboxylated styrene butadiene polymer having a specific gravity of about that of water and a particle 95 size of 0 1 to 0 3 microns.

12 A process for the manufacture of a reagent according to any one of claims 9 to 11, which process comprises reacting an aminoaryl ester or an amino (lower alkyl) 100 amide of a carboxy-substituted barbiturate with a carboxylated latex polymer in the presence of a coupling agent constituted by a water-soluble carbodiimnide.

13 A method of detecting the presence of 105 barbiturates in body fluid, outside the human body, which method comprises observing the inhibition of flocculation caused by said body fluid as compared with a known barbiturate-free body fluid when said 110 first body fluid is added to antiserum against barbiturates and then incubated with a reagent comprising discrete particles of latex polymer having carboxyl groups covalently bonded through an amide linkage 115 to the amino group of an aminoaryl ester or 1 566 014 amino-(lower alkyl) amide of a carboxysubstituted barbiturate.

14 A method according to claim 13, wherein the barbiturate detected is secobarbital, pentobarbital, butabarbital, amobarbital, phenobarbital or barbital.

A method according to any one of claims 13 and 14 wherein the body fluid is urine.

16 A method according to any one of claims 13 and 14 wherein the body fluid is serum.

17 A method according to any one of claims 13 and 14 wherein the body fluid is saliva 15 18 A method according to any one of claims 13 to 17 wherein the mixture of barbiturate-free body fluid and antiserum against barbiturate is incubated prior to the incubation with the latex polymer 20 For the Applicants, CARPMAELS & RANSFORD, Chartered Patent Agents, 43, Bloomsbury Square, LONDON WC 1 A 2 RA.

Printed for Her Majesty's Stationery Office by The Tweeddale Press Ltd, Berwick-upon-Tweed, 1980 Published at the Patent Office, 25 Southampton Buildings, London, WC 2 A l AY, from which copies may be obtained